Patrícia Cavaco-Silva

Clinical and Bacteriological Survey of Diabetic Foot Infections in Lisbon

AIMS An epidemiological survey of diabetic foot infections (DFIs) in Lisbon, stratifying the bacterial profile based on patient demographical data, diabetic foot characteristics (PEDIS classification), ulcer duration and antibiotic therapy. METHODS A transversal observational multicenter study, with clinical data collection using a structured questionnaire and microbiological products (aspirates, biopsies or swabs collected using the Levine method) of clinically infected foot ulcers of patients with diabetes mellitus (DM). RESULTS Forty-nine hospitalized and ambulatory patients were enrolled in this study, and 147 microbial isolates were cultured. Staphylococcus was the main genus identified, and methicillin-resistant Staphylococcus aureus (MRSA) was present in 24.5% of total cases. In the clinical samples collected from patients undergoing antibiotic therapy, 93% of the antibiotic regimens were considered inadequate based on the antibiotic susceptibility test results. The average duration of an ulcer with any isolated multi-drug resistant (MDR) organism was 29 days, and previous treatment with fluoroquinolones was statistically associated with multi-drug resistance. CONCLUSIONS Staphylococcus aureus was the most common cause of DFIs in our area. Prevalence and precocity of MDR organisms, namely MRSA, were high and were probably related to previous indiscriminate antibiotic use. Clinicians should avoid fluoroquinolones and more frequently consider the use of empirical anti-MRSA therapy.

Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Due to their bacterial lytic action, bacteriophage endolysins have recently gained great attention as a potential alternative to antibiotics in the combat of Gram-positive pathogenic bacteria, particularly those displaying multidrug resistance. However, large-scale production and purification of endolysins is frequently impaired due to their low solubility. In addition, a large number of endolysins appear to exhibit reduced lytic efficacy when compared with their action during phage infection. Here, we took advantage of the high solubility of two recently characterized enterococcal endolysins to construct chimeras targeting Staphylococcus aureus. The putative cell wall binding domain of these endolysins was substituted by that of a staphylococcal endolysin that showed poor solubility. Under appropriate conditions the resulting chimeras presented the high solubility of the parental enterococcal endolysins. In addition, they proved to be broadly active against a collection of the most relevant methicillin-resistant S. aureus epidemic clones and against other Gram-positive pathogens. Thus, fusion of endolysin domains of heterologous origin seems to be a suitable approach to design new potent endolysins with changed and/or extended lytic spectrum that are amenable to large-scale production.

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.

Baseline susceptibility of primary HIV-2 to entry inhibitors.

BACKGROUND The baseline susceptibility of primary HIV-2 to maraviroc (MVC) and other entry inhibitors is currently unknown. METHODS The susceptibility of 19 HIV-2 isolates obtained from asymptomatic and AIDS patients and seven HIV-1 clinical isolates to the fusion inhibitors enfuvirtide (ENF) and T-1249, and to the coreceptor antagonists AMD3100, TAK-779 and MVC, was measured using a TZM-bl cell-based assay. The 50% inhibitory concentration (IC(50)), 90% inhibitory concentration (IC(90)) and dose-response curve slopes were determined for each drug. RESULTS ENF and T-1249 were significantly less active on HIV-2 than on HIV-1 (211- and 2-fold, respectively). AMD3100 and TAK-779 inhibited HIV-2 and HIV-1 CXCR4 tropic (X4) and CCR5 tropic (R5) variants with similar IC(50) and IC(90) values. MVC, however, inhibited the replication of R5 HIV-2 variants with significantly higher IC(90) values (42.7 versus 9.7 nM; P<0.0001) and lower slope values (0.7 versus 1.3; P<0.0001) than HIV-1. HIV-2 R5 variants derived from AIDS patients were significantly less sensitive to MVC than variants from asymptomatic patients, this being inversely correlated with the absolute number of CD4(+) T-cells. CONCLUSIONS T-1249 is a potent inhibitor of HIV-2 replication indicating that new fusion inhibitors might be useful to treat HIV-2 infection. Coreceptor antagonists TAK-779 and AMD3100 are also potent inhibitors of HIV-2 replication. The reduced sensitivity of R5 variants to MVC, especially in severely immunodeficient patients, indicates that the treatment of HIV-2-infected patients with MVC might require higher dosages than those used in HIV-1 patients, and should be adjusted to the disease stage.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

Within-lineage variability of ST131 Escherichia coli isolates from humans and companion animals in the south of Europe

Supplementary data at JAC Online). An additional promoter was predicted by Softberry BPROM promoter prediction and consisted of the 235 box of IS18 and a 210 box separated by 13 bp (Figure S1, available as Supplementary data at JAC Online). 10 Interestingly , the TTCAAT 235 box identical to that from Baz (bla OXA-228) was adjacent to the left inverted repeat. OXA-228-like expression in isolate KH243 was compared with that in carbapenem-susceptible A. bereziniae isolate G3-59 by semi-quantitative RT–PCR (qRT– PCR) using rpoB as the reference gene. The primers used for qRT –PCR are shown in Table 1. Three independent experiments were performed using freshly prepared RNA and cDNA and revealed a 56-fold (+3.84) overexpression of bla OXA-228-like in isolate KH243 compared with that in G3-59. To investigate the potential to mediate carbapenem resistance, IS18::bla OXA-257 was cloned into the shuttle vector pWH1266, but we were unable to transfer this into A. bereziniae G3-59. However, the construct was successfully transferred into A. baumannii ATCC 17978 byelectroporation, as previously described for Pseudomonas aeruginosa. 11 Expression of bla OXA-257 in A. baumannii ATCC 17978 raised both imipenem and meropenem MICs from 0.25 to .32 mg/L, demonstrating that IS18::bla OXA-257 is able to confer carbapenem resistance. In conclusion, this study has detected an IS upstream of the intrinsic bla OXA in A. bereziniae, a phenomenon that has not been described in this species so far. IS18 conferred overexpression of OXA-257, which mediated carbapenem resistance in A. bereziniae and A. baumannii. Moreover, because IS18 has previously been described adjacent to acquired bla OXA in A. baumannii, 8 these data suggest a potential for dissemination of OXA-257 in the genus Acinetobacter. 4 Gundi VA, Dijkshoorn L, Burignat S et al. Validation of partial rpoB gene sequence analysis for the identification of clinically important and emerging Acinetobacter species. the Acinetobacter-derived cephalosporinase, carbapenem-hydrolysing oxacillinase and metallo-b-lactamase genes, and of common insertion sequences, in epidemic clones of Acinetobacter baumannii from Spain. Identification of a novel insertion sequence element associated with carbapenem resistance and the development of fluoroquinolone resistance in Acinetobacter radio-resistens. A 10 min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. Research letters 271 JAC Sir, A multiresistant CTX-M-15-producing clonal group of Escherichia coli isolates belonging to phylogroup B2, namely O25b:H4/ ST131, has recently emerged and spread across three continents ; it predominantly causes community-onset infections …

Evolution of the human immunodeficiency virus type 2 envelope in the first years of infection is associated with the dynamics of the neutralizing antibody response

BackgroundDifferently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth.ResultsCD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies.ConclusionOur data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Characterization of multidrug-resistant diabetic foot ulcer enterococci

INTRODUCTION Diabetes mellitus is a highly prevalent chronic progressive disease with complications that include diabetic-foot ulcers. METHODS Enterococci isolated from diabetic-foot infections were identified, evaluated by macro-restriction analysis, and screened for virulence traits and antimicrobial resistance. RESULTS All isolates were considered multidrug-resistant, cytolysin and gelatinase producers, and the majority also demonstrated the ability to produce biofilms. CONCLUSIONS These results indicate the importance of enterococci in diabetic-foot infection development and persistence, especially regarding their biofilm-forming ability and resistance to clinically relevant antibiotics.

Evaluation of the diagnostic performance of the rapid test VIKIA HIV1/2 in a highly complex HIV-1 epidemic

The rapid test VIKIA HIV1/2 was evaluated in 210 Angolan subjects infected with multiple HIV-1 subtypes and complex recombinant forms and 225 seronegative individuals. All infected subjects tested positive (100% sensitivity); all seronegative subjects tested negative (100% specificity). VIKIA HIV1/2 is highly specific and sensitive even in highly complex epidemics.

Prevention of ventilator-associated pneumonia

Invasive mechanical ventilation (IMV) represents a risk factor for the development of ventilator-associated pneumonia (VAP), which develops at least 48h after admission in patients ventilated through tracheostomy or endotracheal intubation. VAP is the most frequent intensive-care-unit (ICU)-acquired infection among patients receiving IMV. It contributes to an increase in hospital mortality, duration of MV and ICU and length of hospital stay. Therefore, it worsens the condition of the critical patient and increases the total cost of hospitalization. The introduction of preventive measures has become imperative, to ensure control and to reduce the incidence of VAP. Preventive measures focus on modifiable risk factors, mediated by non-pharmacological and pharmacological evidence based strategies recommended by guidelines. These measures are intended to reduce the risk associated with endotracheal intubation and to prevent microaspiration of pathogens to the lower airways.